The membrane proximal external region (MPER) of HIV gp41 comprises a highly conserved region involved in HIV viral fusion. It is an important target of antibody (Ab)-mediated neutralization as it contains epitopes for two broadly (b) neutralizing (Nt) monoclonal (M) Abs (2F5 and 4E10) and two MAbs that neutralize a significant, but not truly broad, range of HIV isolates (Z13 and m66.6), making the MPER an obvious target for an AIDS vaccine. However, all attempts to produce an MPER-targeting vaccine have failed. Most of these vaccines have been of three types: (i) synthetic peptides; (ii) MPER sequence grafted onto protein scaffolds; or (iii) onto proteins displayed on virus-like particles. In explaiing these failures, we hypothesize that previous vaccines have not faithfully mimicked the neutralization competent structure (NCS) of the MPER. Our preliminary work has shown that the gp41 transmembrane domain (TMD) is required for the full exposure of the MPER, since its replacement with the TMD from another membrane protein, decreases binding by MAb 4E10. Drs. Scott and Lu previously collaborated on producing a DNA vaccine that expresses a gp41 fragment comprising the MPER and TMD, so as to present the MPER in the context of the cell membrane. However, repeated immunization of rabbits with this DNA vaccine elicited low titer Abs that cross-reacted weakly with MPER peptides and did not neutralize virus; boosting immunizations with a virus-like particle vaccine did not improve anti-MPER titers, probably because of low MPER copy number. We propose to design more effective protein-boost immunogens that will mimic the NCS. In specific aim 1 we plan to design liposome and nanoparticle immunogens that present the gp41 MPER+TMD in high copy number in lipid bilayers, so as to better mimic the NCS of the MPER without added proteins that might distract the Ab response. We will produce DNA, liposome and nanoparticle candidates that have optimized the: (i) MPER, (ii) TMD, and (iii) composition of the lipid environment, based on relative binding affinity by bNt MAbs, the absence of binding by non-Nt mutants of the bNt MAbs, their behavior in a novel membrane leakage assay, and structural stabilization. In specific aim 2 optimized DNA, liposome and nanoparticle candidates will be produced and tested for their ability to elicit MPER-binding activities and Nt Abs. The optimized liposome and nanoparticle vaccines will then be tested as protein boosts (i.e., following DNA priming) with the goal of maximizing MPER-specific Ab titers and Nt potency and breadth. In addition, antigenicity, immunogenicity and structural data will be used to develop a molecular model of the MPER NCS, which should support future vaccine design.